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Lecture 36: "Genome Analysis"

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Biochemistry 2280A
Mel Usselman

Biochemistry Lecture No. 36: Genome Analysis th Friday November 30 , 2012 Q) Assuming a genome size of 2 million base pairs, how many independent clones would need to be constructed and sequenced, if the average insert size is 2000 base pairs? An absolute minimum of 1000 clones would be needed because to be sure you have full coverage you want a library with an excess of several fold (about 20). Creating A Genetic DNA Library For H. Influenza: -Starting with millions of cells, you extract the DNA from H. influenza and sonicate it (instead of restriction enzymes) into random DNA fragments of various sizes. You then purify the fragments through gel electrophoresis and select for those fragments around 2000 base pairs in size. By cloning these 2000 base pair fragments, you receive 20,000 clones (20-fold excess), where each clones represents and independent fragments of the genome. Next, you isolate the plasmids from the 20,000 different clones and sequence them through dideoxynucleotide sequencing. The result is 25,000 sequences from 20,000 clones and approximately 12 million base pairs of sequence altogether (lots of redundancy present). The computer then searches for overlaps between all the sequencing runs by obtaining the end-sequences of the DNA inserts (each one being around 500 base pairs in length). Using this information, it constructs sequence contigs (of which there were 140 in total). If the genome analysis is complete, then only one contig should be present. The fact that there are 140 contigs means that there are 140 gaps (these gaps need to be filled in to know the order of the sequence). Contigs: -A sequence contig is a contiguous DNA sequence representing a portion of the genome after alignment. Sequence assembly is first done by computer searching, looking for overlaps (contigs are not physical entities per se, but is something that the computer organizes). Filling In The Gaps: -There are two kinds of gaps: sequence gaps and physical gaps (they are both resolved differently). Sequence gaps are the ones that can be closed by sequencing clones already present in the library (they are fairly easy to fill in). The computer scans for original clones that span 2 different contigs (5,000 clones that were sequenced on both ends). If you can get a clone that can span two contigs, that means that
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